For the purpose of obtaining the dibasic acid indirect bilirubin in a pure state from the dried canine cholecystic bile, an optimal developing solvent was selected by paper partition chromatography as a preliminary experiment, and it was isolated on cellulose column as an applied experiment. 1. The dibasic acid indirect bilirubin was separable at the starting point in a pure state by paper chromatography under development with the top layer of a n-butanol, acetic acid, water mixture (4:1:5). 2. The dibasic acid indirect bilirubin formed a fixed band at the upper starting place on cellulose column under development with the top layer of a n-butanol, acetic acid, water mixture (4:1:5), and no other substance could be detected there. 3. The dibasic acid indirect bilirubin existing in the fixed band could be eluted out into chloroform with a 1% acetic acid solution. An orange yellow powder was obtained from the eluate by evaporating the solvent in vacuo. 4. Thus separated orange yellow powder agreed well with the crystalline bilirubin in the solubility into organic or inorganic solvents and in the spectrochemical characteristics as well as in the chemical properties.</p

Sakamoto, Takeshi

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Studies on bile pigments V. A method to isolate indirect bilirubin

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Acta Medica OkayamaVolume 10, Issue 4 1956 Article 4SEPTEMBER 1956Studies on bile pigments V. A method toisolate indirect bilirubinTakeshi Sakamoto∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on bile pigments V. A method toisolate indirect bilirubin∗Takeshi SakamotoAbstractFor the purpose of obtaining the dibasic acid indirect bilirubin in a pure state from the driedcanine cholecystic bile, an optimal developing solvent was selected by paper partition chromatog-raphy as a preliminary experiment, and it was isolated on cellulose column as an applied exper-iment. 1. The dibasic acid indirect bilirubin was separable at the starting point in a pure stateby paper chromatography under development with the top layer of a n-butanol, acetic acid, watermixture (4:1:5). 2. The dibasic acid indirect bilirubin formed a fixed band at the upper startingplace on cellulose column under development with the top layer of a n-butanol, acetic acid, wa-ter mixture (4:1:5), and no other substance could be detected there. 3. The dibasic acid indirectbilirubin existing in the fixed band could be eluted out into chloroform with a 1% acetic acidsolution. An orange yellow powder was obtained from the eluate by evaporating the solvent invacuo. 4. Thus separated orange yellow powder agreed well with the crystalline bilirubin in thesolubility into organic or inorganic solvents and in the spectrochemical characteristics as well asin the chemical properties.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLActa Med. Okayama 10, 4, 253-260 (1956) .STUDIES ON BILE PIGMENTSV. A METHOD TO ISOLATE INDIRECTBILIRUBINByTakeshi SAKAMOTODepartment of Internal Medicine,OkayamaUniversit'YMedical School(Director: Prof. Dr. K. Yamaoka)Received for Publication May 24, 1956.Several methods have contributed to isolate bilirubin fromnatural materialsl - 7), and the materials availed there were al-most gallstones. These procedures lie in an extraction of bili-rubin into chloroform from gallstones which were preliminarilyhydrolysed with acid and successively washed with alcohol andether to remove the contaminating impure substances. But itis not a comparatively easy matter to get gallstones of the ma-terial in hand, and, on the other hand, there appeared severalmodifications utilizing biles.9 ) or feces10.11) as the material. Thesematerials were continuously washed with ether-alcohol in an acidmedium, and bilirubin left behind in the residue was extractedinto chloroform. A new purifying method of the separated crudebilirubin on alumina column was further reported12).There are, on the other hand, different kinds of bilirubinin natural materials in view of their reactivities towards theEHRLICH'S diazo reagent, but this fact may well be said to bescarcely mentioned in these reports. The author, therefore,attempted to separate the natural bilirubin according to theattitude towards the EHRLICH'S diazo reagent to get somefavorable resultsl:J-15). The present report in this series containsthe further surveys on an isolation of the indirect bilirubin.Experirnental1. Preparation of Materials.Chloroform ex tract of the dried canine cholecystic bile was1Sakamoto: Studies on bile pigments V. A method to isolate indirectProduced by The Berkeley Electronic Press, 1956254 rr. SAKAMOTO :prepared following the procedure of SHIMADA16). The crude in-direct bilirubin was obtained in an effluate on silica gel columnunder development with chloroform (Fig. 1), and the crude bili-rubin was sampled thereafter after having dried in vacuo.-=._ /JA/1I( DROWNij" " liGHT !JROWN~1t~~. ... ;;Fig 1. Column chromatogram of chloro-form extract of canine bileSample: Chloform extract of the driedcanine cholecystic bile.Adsorbent: Silica gel.Stationary phase: Chloroform.Developing solvent: Chloroform.2. Method.An optimal developing solventfor the separating procedure of theindirect bilirubin was preliminarilyselected by paper chromatography,and isolation of the pure indirect bilirubin was undertaken oncellulose column under development with the same solvent.2. 1. Paper Chromatography.The details of the procedure followed that of the precedingreport in this series1M •2. 1. 1. Samples.The crude indirect bilirubin was dissolved in chloroform atthe rate of 1 mg / ml, and the crystalline bilirubin was availedfor a control, which was also introduced to a chloroform solutionin the same concentration as the crude indirect bilirubin.2. 1. 2. Filter Papers.Toyo No. 51 and SCHLEICHER & SCHULL No. 2043 a filterpapers were availed after no preliminary treatment.2. 1. 3. Development.After 0.01 ml of the chloroform solution was spotted atthe starting point, development was carried out at 12°-14°Cby the one-dimensional ascending method.2. 1. 4. Detecting Method of Several Substances.The GMELIN and EHRLICH'S diazo reactions for bilirubin,the SCHLESINGER'S reaction for urobilin, the EHRLICH'S aldehyde2Acta Medica Okayama, Vol. 10 [1956], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss4/4Studies. on Bile. Pig·ments.·255·reaction for urobilinogen, the ammoniacal silver nitrate, ani-line hydrogen phthalate, and benzidine reactions for reducingsubstances, the phosphomolybdic acid, phosphoric acid, andSbCla reactions for bile acid, and the ninhydrine reaction foramino acid were availed here.2.2. Column Chromatography.2. 2. 1. Materials.The crude bilirubin of 10 mg was dissolved in 2 ml ofchloroform.2. 2. 3. . Columns.Five ml of the top layer of a n-butanol, acetic acid, watermixture (4: 1 : 5) wa'3 added to 20 g of "Cellulose Powder A"(Toyo Filter Paper Co.), which had been stored in a dessicator,and the cellulose was packed in a Tswett tube of 1.8 cm in dia-meter after dryness in vacuo. On the other hand, after the samplein a chloroform solution was adsorbed to 1 g of cellulose powderby evaporating the solvent, 0.25ml of the above top layer wasdropped on it and mixed well. Thus treated cellulose was packedfurther on the column after dryness. One g of cellulose which wastreated as in the first was packed furthermore on the column.2.2.3. Development.The same top layer as mentioned above was availed for de-velopment, which was carried out in a dark place at 12"-14cC.2.2.4. Detecting Method of Several Substances.Bilirubin wds identified by the GMELIN and EHRLICH'S diazoreactions, formation of biliverdin by oxidation, detection ofmesobilinogen by reduction with sodium amalgam, and recogni-tion of the absorption maximum in the visible range at 450 m/1.in a chloroform solution. Fluorescence under the ultravioletlight, the posit.ive SCHLESINGER'S reaction, the negative EHRICH'Saldehyde, EHRLICH'S diazo, and GMELIN reactions were used foran identification of urobilin, while negativeness of the GMELINand SCHLESINGER'S reactions and the positive EHRLICH'S aldehy-de reaction was availed for an identification of urobilinogen.The SALKOWSKY'S reaction for cholesterol and the CASNOVA'Sreaction for phosphatide were also availed. Bile acids were de-tected by SbCl a, phosphomolybdic acid, and phosphoric acidon the paper together with the PETTENKOFER reaction in vitro.3Sakamoto: Studies on bile pigments V. A method to isolate indirectProduced by The Berkeley Electronic Press, 1956256 T. SAKAMOTO:Results1. Paper Chromatography.The crude indirect bilirubin, chromatographed on paper bythe top layer of a n-butanol, acetic acid, water mixture (4: 1: 5),gave a greenish brownish ~rellow colour near the front togetherwith an orange yellow colour at the starting point (Fig. 2).LIf;Hr GREENISHBROWNISHYELLOW Fig. 2. Paper chromatogram of crudeindirect bilirubinSample: Crude indirect bilirubin separat-ed from chloroform extract onsilica gel column.Paper: Toyo No. 51.Development: 30 cm run at 12-14°C by one-dimensional ascending method.Stationary phase: Water.Developing solvent: n-Butanol, acetic acid,water mixture (4: 1 : 5)The former had a greenish yellow fluorescence under the ultra-violet light and the fluorescence was accelerated although it didnot change the colour by the SCHLESINGER'S reaction, and it gavethe positive GMELIN and indirect diazo reactions. The pigmentat the starting point grew brown by the benzidine reaction, butit gave neither the ammoniacal silver nitrate reaction nor theninhydrine reaction. It gave both the positive GMELIN and in-direct diazo reactions, but neither fluorescence, the SCHLESINGER'Sreaction, nor the EHRLICH'S aldehyde reaction was given. Thephosphoric acid, phosphomolybdic acid, and Sbela reactions wereall negative there. The pigment was easily dissolved in chloro-form, carbon tetrachloride, carbon disulfide, and a N /10 NaOHsolution. .A. chloroform solution of the pigment was yellow, andno pigment was transferred into water. The chloroform solutionhad the absorption maximum at 450 mp. in the visible rangeand gave the positive indirect diazo reaction, and the hydro-chloric azobilirubin had:a blue colour. The pigment changed intoa green or bluish green colour by the procedures followed that4Acta Medica Okayama, Vol. 10 [1956], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss4/4Studies on Bile Pigments. 257of LEMBERG aridLEGGE!l\ and the pigment changed into a sub-stance which gave the positive EHRLICH'S aldehyde reaction byreduction with sodium amalgam.The chromatogram, developed by a mixture or the top layerof a mixture of n-butanol, ethanol, water (4 : 1 : 2), iSQ-butanol,acetic acid, water (4 : 1 : 5), iso-butanol, ethanol, water (4: 1: 2),or so, had a more or less clear tailing in the frontal region tocompare with the above result, and it had also no partitionatedsubstance between the starting point and the front.The chromatogram, developed by the top layer ofa mixtureof n-butanol, phenol, water (4 : 1 : 2), n-butanol, lutidine, water(4 : 1 : 2), n-butanol, collidiI).e, water (4: 1 : 2), . or n-butanol,ethanol, ammonia, water (40 : 10 : 1 : 49), showed a remarkabletailing at the starting point and the frontal region.2. Column Chromatography.The results of column partition chromatography with cel-lulose powder under development with the top layer of a n..,butanol, acetic acid, water mixture (4 : 1 : 5) is shown in Fig. 3,'LtGNTGRE£N/SHYELLOWORANGE YELLOW,:....:.." "; ..,START ..Fig. 3. Column chromatogram of crudeindirect bilirubinsample: Crude indirect bilirubin.Column: Cellulose powder.Stationary phltse: Top layer of. an-butanol,acetic acid, water mix-ture (4: 1 : 5)Developiug solvent: do.and the crude bilirubin gave two co-loured bands, an orange yellow oneat the upper starting place and ayellowish green one at the frontalplace. The effluate of the latter gavethe positive GMELIN and indirect diazo reactions, andthe.GMELINreactant grew red in several hours. It gave a light yellow fluo-rescence under the ultraviolet light, and it was accelerated bythe addition of the SCHLESINGER'S reagent. It gave a reddishtone very slowly by the addition of the EHRLICH'S aldehydereagent, and the colour turned into green in half a day by let-5Sakamoto: Studies on bile pigments V. A method to isolate indirectProduced by The Berkeley Electronic Press, 1956258 T.~SAKAMOTO :ting it standing. The effluate, furthermore, gave neither -thepositive SALKOWSKY'S nor CASNOVA'S reaction.The column was dried as possible after about 250 ml of thesolvent passed through it, and then elution of the orange yellowband at the starting place with a 1 % acetic acid solution intochloroform gave an orange yellow solution, which was dried invacuo after washing with water to get an orange yellow pow-der.The orange yellow power was well dissolved into chloro-form, carbon tetrachloride, carbon disulfide, lutidine, collidine,dioxane, and aN/ID NaOH solution, and scarcely into alcohol,butnot into water at all. A chloroform solution had the absorptionmaximum at 450 mp, in the visible range, and gave the positiveGMELIN and indirect diazo reactions, but it had no fluorescenceunder the ultraviolet light, and neither the SCHLESINGER'S reac-tion nor the PETTENKOFER reaction was positive. The oxides ofthis pigment by LEMBERG and LEGGE'S method!') was green orgreenish blue, and the reductants by sodium amalgam gave thepositive EHRLICH'S aldehyde reaction.DiscussionWhen the pigment, which was prepared from the caninecholecystic bile on silica gel column under development withchloroform, was partitionated by paper chromatography withthe top layer of a n-butanol, acetic acid, water mixture (4:1: 5),the substance partitionated at the frontal region agreed wellwith that of the effluate on cellulose column with the same dQ-veloping solvent. The pigment remained at the starting poilltin the former also agreed well with the one remained at thestarting place in the latter.That is, both the frontal spot on the paper and the efflua,teon cellulose column gave the positive GMELIN and indirect diazoreactions, and further both of the two had fluorescences und,erultraviolet light and gave the positive SCHLESINGER'S reaction.And then, the main substance existing there will be urobilinthough the indirect bilirubin may be mingled in part. A browncolour near the front on the paper by the SbCl;; reaction maysuggest an existence of a substance like bile acid1o). To compllre6Acta Medica Okayama, Vol. 10 [1956], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss4/4Studies on Bile Pigments. 259the substance remaining at the starting point on the paper withthe one on cellulose column, the former not only gave the posi-tive GMELIN and indirect diazo reactions, but also such reactions.as to detect urobilin, urobilinogen, bile acid,' steroid, reducingsubstance exclusive of the benzidine reaction, or amino acid wereall negative, and the pigment recognized there agreed well withbilirubin itself together with its transferability into orgariic orinorganic solvent, and with its absorption maximum in a: chloro-form solution. And further, the effluate gave the positive. in-direct diazo reaction, of which colour turned into blue, tha-t ofatypical hydrochloric azobilirubin. It was oxidized, into biliver-din and reduced into mesobilinogen by sodium amalgam., rTh.eabove results leave no doubt that the pigment is the dibasic acid'indirect bilirubin alone. The latter pigmentcontained:in theupper fixed band on cellulose column could be obtained. in anorange yellow powder, which was also identified to the verydibasic acid bilirubin in view of the transferability into organicor inorganic solvent, absorption maximum in a chloroform solu-tion, the positive GMELIN and indirect diazo reactions, trans-formation into biliverdin by oxidation or into mesobilinogen byreduction with sodium amalgam and so on. Such substancesother than bilirubin as urobilin, urobilinogen, bile:acid.,stexoid,reducing substance exclusive of the benzidine reaction,ot'aminoacid were all undetectable. These results also leave' rrodoubfthat this band will 'contain the dibasic acid indirect ;bilir-uhj.nalone as in the case of the starting point on the paper.On the basis of these data, the most reasonable conc;'lusi,orlmay be that this method is very easy and simple for~xtra.ctingthe dibasic acid bilirubin from bile, and that it bears compar.isonwith the methods already reported by many authors in view ofan aO'reement of the re~ult and the theory between a pr:erimiria-o 'ry experiment with paper partition chromatography and an ap-plied experiment with column partition chromatography.SummaryFor the purpose of obtaining the dibasic acid indire~t bili-rubin in a pure state from the dried canine cholecystic bile, anoptimal developing solvent was selected by paper partition chro-7Sakamoto: Studies on bile pigments V. A method to isolate indirectProduced by The Berkeley Electronic Press, 1956260 T. SAKAMOTO :matography as a preliminary experiment, and it was isolatedon cellulose column as an applied experiment.1. The dibasic acid indirect bilirubin was separable at thestarting point in a pure state by paper chromatography underdevelopment wi th the top layer of a n-butanol, acetic acid, watermixture (4: 1 : 5).2. The dibasic acid indirect bilirubin formed a fixed bandat the upper starting place on cellulose column under develop-ment with the top layer of a n-butanol, acetic acid, water mix-ture (4 : 1 : 5), and no other substance could be detected there.3. The dibasic acid indirect bilirubin existing in the fixedband could be eluted out into chloroform with a 1 % acetic acidsolution. An orange yellow powder was obtained from the eluateby evaporating the solvent in vacuo.·4. Thus separated orange yellow powder agreed well withthe crystalline bilirubin in the solubility into organic or inorga-nic solvents and in the spectrochemical characteristics as wellas in the chemical properties.References1) STADELER, F. : Ann., 132, 323, 1864. - 2) KUSTER, W.: Z. physioI.Chcm., 94, 136, 1915. - 3) KUSTER, W. : ibid., 99, 86, 1917. - 4) KUSTER, W.if/id., 121, 80, 1922. - 5) KUSTER, W. and R. HAAS: ibid., 141, 279, 1924.-6) FISCHER, H. : ibid., 73, 204, 1911. - 7) FIS::;HER, H. and R. HESS : ibid.,194, 193, 1931. - 8) GIBSON, R. B. and R. C. L")WE: J. BioI. Chem., 123,xli, 1938. - 9) L3MBERG, R. and J. W. LSGcr: Aust. J. Exptl. BioI. Med.Sci., 18, 95, 1940. - 10) FIS::;HER, H. and H. L'30WITZKY: Z. physiol. Chem.,258, 255, 1939. - 11) L')WRY, P. '1'., 1. B')SSENMAIER, and C. J. WATS:JN :.1. BioI. Chem., 202, 305, 1953. - 12) GRAY, C. H. and J. WHIDBORNE:Biochem. J., 40, 81, 1946. - 13) SAKAMOTO, T. : Acta Med. Oknyama, 10,11, 1956. - 14) l::;\KAMOTO, T. : ibid., 10, 30, 1956. - 15) SAKAMOTO. T. :ihid .. 10, 227, 1956. - 16) SHlMADA, Y. : Igaku Kenkyu, 24, 1894, 1954 (ill.J a pancsc).8Acta Medica Okayama, Vol. 10 [1956], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss4/4

Studies on bile pigments V. A method to isolate indirect bilirubin

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